Single-molecule analytical methods can provide insight into biomolecular dynamics, including by extracting characteristics of molecular interactions in complex mixtures where such information could otherwise be lost in ensemble averaging.
The optical confinement generated by it nanoapertures fabricated on a silica substrate can be used to perform single-molecule fluorescence measurements at physiologically relevant background concentrations of fluorophore-labeled biomolecules. For example, zero-mode waveguides (ZMWs), which are sub-wavelength nanoapertures generated in a metal film, can allow observation of single-molecule phenomena. When light is shone through a zero-mode waveguide, photons having wavelengths greater than a threshold value can be prevented from propagating through the waveguide. The remaining evanescent waves can exponentially decay at the glass/water interface of the ZMWs, leading to a very small detection volume near the interface, e.g., on the scale of zeptoliters. Thus, ZMWs can provide improved signal-to-background ratios (SBRs) of single-molecule fluorescence, permitting single fluorophore-labeled biomolecules to be observed in imaging buffers containing physiologically relevant, micromolar concentrations of fluorophore-labeled ligands.
However, such SBR gains of the nanoapertures can be limited to certain concentrations, for example, with fluorophore-labeled biomolecules such as nucleic acids, at concentrations of up to 1 micromolar. Above this concentration of fluorophore-labeled nucleic acids, and at even lower concentrations of fluorophore-labeled proteins, non-specific binding of the fluorophore-labeled biomolecules to the surfaces of the nanoaperture can undermine the SBR gains.
Accordingly, there is a need for nanoapertures with reduced non-specific adsorption of biomolecules.